A recent report by Kohanski et al. demonstrated that multiple classes of bactericidal antibiotics stimulate the production of hydroxyl radicals thus accelerating bacterial cell death in Escherichia coli. Although different classes of antibiotics have unique mechanisms of action, their data suggest that they mediate killing through a common pathway involving hydroxyl radicals. By an unknown mechanism, bactericidal antibiotics transiently increase the reduction of NAD+ via the TCA cycle, increasing superoxide generation in the ETC. Superoxide damages iron-sulfur clusters;the released ferrous ion is thus available for the Fenton reaction generating hydroxyl radicals. Validating these studies, allelic replacement gene knockouts of the TCA cycle enzymes aconitase (acnB) and isocitrate dehydrogenase (icdA) abrogated the killing effect of bactericidal antibiotics. In addition, bactericidal antibiotics did not induce hydroxyl radical formation in the TCA cycle mutants. Data presented in this application demonstrate that 26% of clinical Staphylococcus epidermidis isolates are TCA cycle defective. In addition, oxacillin time-kill studies against a S. epidermidis 1457 aconitase mutant (1457 acnA::tetM) revealed that killing was decreased 2.6 log10 compared to wild type. Therefore, the loss of TCA cycle activity represents a physiological adaptation of the bacterium to an environment where antibiotic pressure is prevalent. The central hypothesis of this application is that antibiotic pressure selects for TCA cycle mutants within the S. epidermidis population. This hypothesis will be tested by first determining if bactericidal antibiotics induce hydroxyl radical formation in S. epidermidis. Secondly, we will determine the percentage of clinically relevant S. epidermidis isolates that are defective in TCA cycle function. Lastly, we will determine if TCA cycle mutants are more recalcitrant to antibiotic therapy using a guinea pig tissue cage model. This proposal will yield significant new information regarding the physiological adaptation of bacteria allowing growth under antibiotic pressure. Novel means to prevent and treat bacterial infections may be suggested as a result of these studies. PUBLIC HEALTH RELEVANCE: Experiments proposed in this application are designed to determine whether the use of certain antibiotics selects for bacteria that are resistant because of mutations in a common metabolic pathway.